Astrocytes during HIV infection of the brain : relevance for neuropathogenesis

Sammanfattning: The overall aim of the present thesis was to characterize the role that astrocytes play for neuropathogenesis of AIDS dementia complex (ADC). We studied whether it is possible to derive the phenotype of HIV-1 virus by analyzing V3 sequences. Our analysis, conducted by comparing V3 sequences and replicative properties of 9 HIV-1 isolates (from four patients), indicated that the shift from non syncytium inducing to syncytium inducing phenotype did not always correlate with an increase of V3 net charge. Thus extrapolation of the biological properties of HIV-1 variants from V3 sequences it is not always possible. The frequency of ADC in cohorts of patients treated with zidovudine (AZT) is very low. We analyzed whether AZT resistant strains could be found in the CSF and blood of six patients treated with AZT. Blood isolates resistant to AZT have been shown to be mutated at positions 41, 67, 70, 215 and 219 of the reverse transcriptase gene. In our study, we could show that in the isolates from 4 (of 6) patients different pattern of mutations were found in CSF isolates as compared to what found at the same time point in blood. These results show that AZT reaches the CSF and give support to the different evolution of the HIV-1 virus in the brain and systemic compartments. In order to further clarify the role of astrocytes in HIV-1 neuropathogenesis, we infected primary fetal astrocytes with 26 primary HIV-1 isolates with defined biological properties, including isolates with slow/low and rapid/high phenotypes and different chemokine receptor usage. We demonstrated that human fetal astrocytes could be infected in vitro with primary HIV-1 isolates independently of their biological phenotype and chemokine receptor usage. Viral replication was not sustained and stimulation with the cytokine IL-1[beta] promoted virus production in astrocytes. The HIV-1 receptor(s) on astrocytes was studied by analysing CD4 expression and chemokine receptor mRNAs and protein expression in primary astrocytes. We could amplify the mRNAs for CXCR4, CXCR2, CCR5, CCR3, CCR2 and the orphan receptors APJ and Bonzo/STRL33/TYMSTR, although the astrocytes did not express any of these receptors (nor CD4) at the cell surface. This result was confirmed when the presence of chemokines RANTES, MIP-1[alpha] MIP-1[beta], MCP-1 and SDF-1[beta] in culture did not induce mobilization of intracellular calcium influx in astrocytes. Therefore we suggest that primary HIV-1 isolates infect astrocyte independently of CD4 and chemokine receptors. Since neuropathological studies conducted on AIDS brains have shown that astrocytes are activated and undergo apoptosis, we focused our attention on molecules involved in the regulation of apoptosis. We analyzed the expression of Fas and Fas ligand (FasL) in brain tissues obtained from HIV-1 infected subjects, including asymptomatic HIV-1 carriers and AIDS patients, and controls. The amount of Fas transcripts amplified from the AIDS brains was higher than from the brain of asymptomatic carriers. The FasL transcripts were expressed in the brain of patients who had developed AIDS and of one asymptomatic carrier. Fas was predominantly expressed by reactive astrocytes and FasL by CD3+ T-cells located in proximity of reactive astrocytes. These results indicate that the Fas/FasL pathway may be involved in apoptosis dysregulation in the brain during AIDS. This possibility was reinforced by the interesting finding that the levels of soluble Fas (sFas) and sFasL were significantly higher in the CSF of patients with ADC (n=29) as compared to ADC negative patients (n=22) and HIV-1 negative controls (n=39). A decline of CSF sFas levels was observed in patients treated with highly active antiretroviral therapy (HAART) in parallel to the reduction of viral load in CSF. Taken together, theses results suggest that sFas and sFasL may represent useful markers for ADC diagnosis and for monitoring immune activation in the brain during HAART. We also explored the possibility that upregulation of Fas expression during HIV-1 infection would render these cells susceptible to bystander cell killings executed through virus-specific cytotoxic T-cells (CTLs) invading the brain. Bystander cell killing has been shown to occur through the Fas-Fas L pathway in different experimental conditions. The response of a panel of astroglioma cell lines and primary human astrocytes to supernatants from peptide activated CTLs was studied. Our results showed that virus specific CTLs can produce soluble factors capable of inducing apoptosis in 5110 astroglioma cell lines., Fas L is not the only factor involved in this pathway of cell damage. Our results showed that in the supernatant from activated CTLs are present additional factor(s) that can mediate the apoptosis of astroglioma lines, alone or in synergy with TNF-[alpha]